JP2001524823A - Novel adenovirus vector that can facilitate transgene persistence enhancement - Google Patents

Abstract

  (57) [Summary] The present invention relates to a novel transgene capable of promoting the sustained expression of a transgene delivered by a vector into a cell. This vector is an E1 / partial E3 deletion vector containing a transgene operably linked to an expression control sequence, preferably a CMV promoter. The present invention also relates to a composition comprising the adenovirus vector of the present invention, and a method for sustained expression of a transgene in cells of an individual by administration of the composition.

Description

DETAILED DESCRIPTION OF THE INVENTION              Can facilitate transgene persistence                      New adenovirus vectorsIntroduction   The present invention allows for the sustained expression of a transgene delivered by a vector into a cell. Novel adenovirus vectors that can be facilitated. These The vector is a vector operably linked to an expression control sequence, preferably a CMV promoter. An E1 / partial E3 deletion vector containing the transgene. The present invention also relates to the present invention. A composition comprising the defined adenovirus vector and administering the composition. Thus, the present invention also relates to a method for producing sustained expression of a transgene in an individual cell.Background of the Invention   Adenoviruses are adequate with standard genetic and molecular biology studies Non-enveloped nuclear DNA having a genome of about 36 kb, characterized by Illus (Horwitz, MS, "Adenoviridae an d Their Replication ", Virology, 2nd edition, Fi elds et al., Raven Press, New York, 1990). Two Early (known as E1-E4) the development of transient types of viral proteins. ) And late (L1-L5) transcription units. Between these events The border is viral DNA replication.   Recombinant adenovirus has an affinity for both dividing and non-dividing cells, with minimal Pathogenicity, ability to replicate to high titers for vector stock production, and large Several, including the possibility of having an insert, for use as a gene transfer vector (Berkner, KL, Curr. Top. Micro). . Immumol. 158: 39-66, 1992; Jolly, D .; , Can cer Gene Ther. 1: 51-64, 1994).   The adenovirus cloning capacity depends on the amount of adenovirus present in the vector. It is proportional to the size of the genome. For example, a cloning capacity of about 8 kb would Deletion of certain regions of the viral genome that are not important for length, eg, E3 293 cells (Graham, FL, J. Gen. Virol) . 36: 59-72, 1977) or A549 cells (Imler et al., Gene). Ther. 3: 75-84, 1996), which can be repaired during transport, e.g. It can result from deletion of a genomic region such as 1. Such an E1-deleted vector replicates Become replication-defective. Upper limit of vector DNA capacity is wild type About 105% to 108% of the length of the genome. Further considerations in vector design Cell lines in which denovirus genomic modifications supply other viral gene products during transport For example, the complementation of E2a is possible (Zhou et al., J. Virol. 7). 0: 7030-7038, 1996), the complement of E4 (Kroughliak et al., H um. Gene Ther. 6: 1575-1586, 1995; Wang et al., Gene Ther. 2: 775-783, 1995) or of protein IX Complementation (Caravoxyri et al., J. Virol. 69: 6627-6633; Krugliak et al., Hum. Gene Ther. 6: 1575-1586 , 1995). The maximum carrying capacity is Can be obtained using an adenovirus vector lacking the coding sequence. (Kochan et al., Proc. Natl. Acad. Sci. USA) 93: 5731-5736, 1996; Fisher et al., Virology 2. 17: 11-22, 1996).   The transgenes so far expressed by adenovirus vectors are p 53 (Wills et al., Hum. Gene Ther. 5: 1079-188, 1). 994); dystrophin (Vincent et al., Nature Genetic). s 5: 130-134, 1993); erythropoietin (Descamps) Hum. Gene Ther. 5: 979-985, 1994); Ornichi Transcarbamylase (Stratford-Perricaudet et al., Hum. Gene Ther. 1: 241-256, 1990; We et al. B iol. Chem. 271, 3639-3646, 1996); adenosine dea Minase (Mitani et al., Hum. Gene Ther. 5: 941-948). Interleukin-2 (Haddada et al., Hum. Gene.). Ther. 4: 703-711,19 93); α1-antitrypsin (Jaffe et al., Nature Geneti). cs 1: 372-378, 1992); thrombopoietin (Ohwada et al.) , Blood 88: 778-784, 1996); and cytosine deaminase. (Ohwada et al., Hum. Gene Ther. 7: 1567-1576, 1). 996).   Adenovirus affinity for airway cells is the most common in Caucasians Adenovirus for cystic fibrosis (CF), a novel autosomal recessive disorder Have a special connection to the use of Blocks cAMP-regulated C1 channels in airway epithelium Harmful cystic fibrosis transmembrane conductance regulation (CFTR) inheritance Child mutation results in lung dysfunction (Zabner et al., Nature Genet) ics 6: 75-83, 1994). Modified to include the CFTR gene Adenovirus vectors have been developed (Rich et al., Hum. Gene. Ther. 4: 461-476, 1993), studies indicate that these vectors are associated with CF disease. In the nasal epithelium of the elderly (Zabner et al., Cell 75: 207-216, 1993). ), In rat and primate airway epithelium (Zabner et al., Nature G). enetics 6: 75-83, 1994), and in the airway epithelium of CF patients (C crystal et al., Nature Genetics 8: 42-51, 1994. ) Indicates that the CFTR can be sent. Recent research has shown that CFTR Adenovirus vector containing a DNA sequence encoding a CF patient's airway Administering to the epithelial cells can provide a functional chloride ion channel for the treated epithelial cells. Can be repaired (Zabner et al., J. Clin. Invest. 97: 1504-1511, 1996).   The use of adenovirus vectors in gene transfer studies to date has often been If it is temporary. At least in part, this limitation is due to antigen Is expressed by and destroys or adversely affects adenovirus-infected cells. Host immune response to a viral protein that triggers a potentially pathological inflammatory response. (Yang et al., J. Virol. 69: 2004-2015, 1995). Yang et al., Proc. Natl. Acad. Sci. USA 91: 440 7-4411, 1994; Zsengeller et al., Hum. Gene Ther. 6: 457-467, 1995; Worgall et al., Hum. Gen e Ther. 8: 37-44, 1997; Kaplan et al., Hum. Gene   Ther. 8: 45-56, 1997). Host against adenovirus infection In particular, the immune response caused by cytotoxic T cells that lyse infected cells and display viral antigens -Generation of lymphocytes (CTL), virus-infected cells by tumor necrosis factor (TNE) Cell lysis, interferon synthesis, apoptosis induction, antibody production, and Involves other immune mechanisms (Smith, GL, Trends Microb) iol. 2: 81-88, 1994). Adenovirus is integrated into the cell genome Since no host immune response destroys virions or infected cells, adenovirus Has the potential to limit gene delivery based on Adenovirus immune response Has significant obstacles to high dose or repeated administration of denovirus vector (Crystal, R., Science 270: 404-410, 1995) ).   Various measures to avoid host immune responses that limit the persistence of transgene expression Are generally used to modulate or weaken the immune response itself. Requires engineering of the vector.   Administration of an immunosuppressant with an adenovirus vector can It has been shown to extend the duration (Fang et al., Hum. Gene Ther. 6). : 1039-1044, 1995; Kay et al., Nature Genetics.   11: 191-197, 1995; Zsellanger et al., Hum. Gen e Ther. 6: 457-467, 1995).   Inject adenovirus vectors with alternative serotypes Has been found to avoid host immune responses to some extent (Mack Hum. Gene Ther. 8: 99-109, 1997). Animal model Studies show that persistence of transgene expression can vary between different mouse strains (Barr et al., Gene Ther. 2: 151-155, 1995). .   The lack of sustained expression of the adenovirus vector delivery transgene is Limitations caused by selection of promoters or transgenes contained in the transcription unit (Guo et al., Gene Ther. 3: 802-801, 19). 96; Tripathy et al., Nature Med. 2: 545-550,19 96).   Adenovirus contained in a recombinant vector to weaken the host immune response Modifications to genomic sequences have been attempted (Yang et al., Nature Genet). ics 7: 362-369, 1994; Lieber et al. Virol. 7 0: 8944-8960, 1996; Gorziglia et al. Virol. 70: 4173-4178; Kochanek et al., Proc. Natl. Aca d. Sci. USA 93: 5731-5736, 1996; Fisher et al. Virology 217: 11-22, 1996).   In addition to the deletion of the adenovirus E1 region, the first generation adenovirus vector To increase vector packaging capacity and reduce viral gene expression Three regions often contain modifications (Yang et al., J. Vrol. 69: 200). 4-2015, 1995; Zsellanger et al., Hum. Gene The r. 6: 457-467, 1995; Brody et al., Hum. Gene The r. 5: 821-826, 1994). However, the adenovirus E3 region Contains certain proteins that regulate the primary antiviral immune response. E3 transcription The unit is 12.5K, 6.7K, gp19K, 11.6K, 10.4K, 14.5. K and 14.7K proteins (Wold et al., Trends Mic) robotial. 2: 437-443, 1994). E3 14.7K, 14.5 K and 10.4K proteins protect infected cells from TNF-induced cell lysis Can be. Adenovirus E3gp19K protein is activated by MHC class I antigen. And retain them in the endoplasmic reticulum, which are cytotoxic T-lymphocytes. (CTL) to prevent cell surface presentation and killing of infected cells (Wo ld et al., Trends Microbial. 2: 437-443, 1994) Suggests that its presence in a recombinant adenovirus vector could be beneficial You. E3 11.6K gene (adenovirus d) eath protein)) is required for cell lysis and adenovirus release from infected cells (Tollefson et al., J. Virol. 70: 229). 6-2306, 1996; Tollefson et al., Virology 220: 152-162, 1996).   Previous designs of E3 region modified adenovirus vectors have been used in test animal Only transient expression of the transgene in the lung is shown (Yang et al., J. Vr. ol. 69: 2004-2015; Zsellanger et al., Hum. Gene   Ther. 6: 457-467, 1995). Reduces viral gene expression Adenovirus in adenovirus vector to further increase retention capacity A modification to the E4 region has been introduced (Armentano et al., Hum. Gen. e Ther. 6: 1343-1353, 1995). But adenovirus ・ Experiments in which the vector was introduced into nude mice, especially using the CMV promoter When controlling transgene expression, the environment of the adenovirus E4 genomic region Demonstrated to be a determinant in current persistence (Kaplan et al., Hum. G. ene Ther. 8: 45-56, 1997; Armentano et al. V irol. 71: 2408-2416, 1997).   The current status of adenovirus vector-based gene delivery is Development of novel adenovirus vectors that demonstrate the ability to sustain and sustain expression of offspring Need.Summary of the Invention   The present invention promotes sustained expression of a transgene that is delivered by the gene into the cell And a novel adenovirus vector. This vector Is a transgene operably linked to an expression control sequence, preferably a CMV promoter Is an E1 / partial E3 deletion vector. The present invention also relates to the present invention. A composition comprising a virus vector, and administering the composition to cells of an individual. It also relates to a method for producing sustained expression of a transgene.BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows a schematic diagram of pAd / E4 + / E3 △ 1.6.   FIG. 2 shows a schematic diagram of the Ad2 / CFTR-16 vector.   FIG. 3 shows a schematic of E3 modifications to the Ad2 / CFTR-16 vector.   FIG. 4. Ad in the lungs of immunoreactive and nude C57B1 / 6 mice. 2 shows expression of human CFTR from the 2 / CFTR-16 vector.   Figure 5. Ad2 / CFT in the lungs of immunoreactive C57B1 / 6 mice. Figure 4 shows expression of human CFTR from R-16 vector.   FIG. 6 shows Ad2 / CFTR in the lungs of immunoreactive BALB / c mice. Figure 3 shows expression of human CFTR from the -16 vector.   FIG. 7 shows Ad2 / CFTR in the lungs of immunoreactive BALB / c mice. Figure 3 shows expression of human CFTR from the -16 vector.   FIG. 8 shows Ad2 / CFTR-16 in the lungs of immunoreactive C3H mice. Figure 4 shows expression of human CFTR from vector.   FIG. 9 shows the percentage of hCFTR expression compared to endogenous mCFTR expression in mice. Shows the analysis.   FIG. 10 shows that in treated and untreated CFTR mutant cells (A) Comparison between the base potential difference and (b) hyperpolarization.   FIG. 11 shows vector administration in C57B1 / 6 mice and BALB / c mice. 2 shows the CTL response to   FIG. 12 shows transgene expression in BALB / c mice after repeated administration.Detailed description of the invention   The present invention relates to expression control sequences, preferably cytomegavirus immediate early (CMV) promoters. Can promote sustained expression of a transgene operably linked to a promoter It relates to a novel adenovirus vector. These vectors contain the length of the transgene By generating expression for a period of time, the vector can be inverted to obtain a particular phenotypic result. This is advantageous in reducing the need for re-administration. The adenovirus vector of the present invention contains all or a part of the E1 region and all of the E3 region. Or the adenovirus genome deleted in part. Adenovirus E The four regions are preferably carried in a vector and driven under the control of a CMV promoter. Prolong the duration of expression from the transgene.   The adenovirus vector of the present invention is preferably replication deficient, for example, These are deleted for genes that are important for viral self-replication. This Deletions are safer for administration to individuals, and It has the additional advantage of being able to have NA inserts. Favorable fruit In one embodiment, the vector of the invention contains the E1A protein required for autonomous replication. Remove coding sequence and remove all or part of E1B region of viral genome Contains a deletion of the E1 genomic region of the virus.   Protein in the adenovirus genome to optimize packaging capacity The quality IX coding sequence can be carried in a vector of the invention. The present invention In a particular embodiment, the protein IX coding sequence is located at the boundary of the E1 sequence. From those positions in the adenovirus genome to, for example, the virus Can be relocated into the E4 region of the Such a rearrangement is Can generate replication-competent adenovirus (RCA) during vector production Can mediate recombination with homologous adenovirus sequences in packaging cell lines (Hehir et al., J. Virol. 70: 8459-8467, 1996).   The adenovirus vector of the present invention contains all or all of the adenovirus E2 genomic region. Owns a part.   The adenovirus vector of the present invention contains all or all of the adenovirus E4 genomic region. Owns a part. Such a sequence may be transgenes under the control of the CMV promoter. When contained in an offspring-containing adenovirus vector, Appear to promote the persistence of transgene expression in plants (Armentano et al. J. Virol. 71: 2408-2416, 1997). Preferably, The Ming adenovirus vector is used to promote E. coli transgene expression. It carries the coding sequence of the 4ORF3 gene.   The adenovirus vector of the present invention contains all or all of the adenovirus E3 genomic region. Owns a part. For example, truncation of the E3 coding sequence or Modifications to the E3 region, such as deletions that remove certain open reading frames Decoration is possible as long as these changes do not interfere with the sustained expression of the transgene. Modification to E3 region prevents gp19K protein gene from displaying viral antigen And thereby limit the CTL response to adenovirus-infected cells Preferably for its specific immunomodulatory role in (Wold etc, Trends Microbiol. 437-443, 1994). For example, 10 . Genes for other immunomodulatory proteins, such as 4K, 14.5K and 14.7K Is also within the modifications considered for the E3 region.   In a preferred embodiment of the invention, E3 is contained in the adenovirus vector of the invention. The gene for the 11.6K protein is not retained. This protein is adenovirus Is not involved in the lysis of infected cells (Tollefson et al., J. Virol. 70: 2296-2306, 1996; Tollefson et al., Virlog. y 220: 152-162, 1996). E3 open other than 11.6K gene Overexpression of the reading frame also reduces the effect of 11.6K gene expression be able to.   In a preferred embodiment of the invention, the adenovirus vector is Ad2 / CFT. R-16, which is a CMV prototype to which the transgene can be functionally linked. It contains a motor, plus an E1 deletion and a 1.6 kb partial deletion from the E3 region Is also contained. This is because the transgene and its expression control sequence can be inserted. A replication-defective vector containing a deletion in the E1 region Preferably, an MV promoter is included. This is even more wild type adenowi It contains the Luz E2 and E4 regions. This vector contains the adenovirus E3 region comprising 1549 nucleotides from nucleotides 29292 to 30840 (Roberts, RJ, et al., Adenovirus DN). A, in Developments in Molecular Virol ogy, W.C. Doerfler, 8: 1-51, 1986). Vector Ad Modifications to the E3 region in 2 / CMV / E3 △ 1.6 result in: (a) E3 region All downstream splice receptor sites in the region are deleted, and only mRNA (a) is -Is thought to be synthesized from a motor (Tollefson et al., J. Viro l. 70: 2296-2306, 1996; Tollefson et al., Virol. (Ogy 220: 152-162, 1996); (b) E3A poly A site deleted But retains the E3B poly A site; (c) E3gp19K (MH (C1 binding protein) is retained; and (d) E3 11.6K (Ad Gene has been deleted.   The adenovirus vector of the present invention includes, but is not limited to, an adenovirus serotype. And any other adeno, including all other preferably non-neoplastic serotypes Adenovirus genomic sequence from the serotype of serotype can be used .   The adenovirus vector of the invention allows for sustained expression of the transgene It may contain a transgene present in the transcription unit. Adenowi of the present invention Luss vector sustained transgene is a long-term sustained level of transgene. Are defined as those that result in the expression of   The transgene herein is any non-native to the adenovirus genome. Is defined as   The transcription unit of the adenovirus vector of the present invention is referred to herein as a transgene. And an expression control sequence such as a promoter or enhancer, And other tasks that can be used to regulate or prolong expression. The desired regulatory elements (all of which are functionally (To be ligated). Sustained transgene release The use of any expression control sequence to facilitate expression is within the scope of the invention. like this The sequence or element can result in tissue-specific expression, or Can be induced by stimulation.   Preferably, the cytomegalovirus (CMV) immediate early promoter (Bash Art et al., Cell 41: 521-530, 1985). Ends of this promoter that control or function similarly in the transgene Truncated fragments can be used. The CMV promoter in the transcription unit It is located 5 'to the transgene. They lead part of the full-length promoter Whether or not it allows for sustained expression of the transgene is tested using the assays described below. Can be In a preferred embodiment, nucleotides -523 to -14 CMV promoter region is used in the adenovirus vector of the present invention.   Polyadenylation can be located at the 3 'end of the transgene in the transcription unit Signals are derived from, but not limited to, bovine growth hormone (BGH) and SV40 Includes a polyadenylation signal.   The transcription unit is delivered and expressed in the adenovirus vector of the invention. Transgenes that can be used include, but are not limited to, enzymes, blood derivatives, hormones, Lymphokines such as interleukins and interferons, coagulation factors, Long hormones, neurotransmitters, tumor suppressors, apolipoproteins, antigens and anti- Includes transgenes encoding the body and other biologically active proteins. Book Specific transgenes that can be encoded by the transcription units of the invention include, but are not limited to, Cystic fibrosis transmembrane regulator (CFTR), dystrophin , Glucocerebrosidase, tumor necrosis factor, p53, retinoblastoma (Rb ), And adenosine deaminase (ADA). Antisense molecule or Transgenes encoding ribozymes are also within the scope of the invention.   The transcription unit is capable of expressing the transgene in the adenovirus vector of the invention. It can be inserted at any given site. The transcription unit is Preferably, it is inserted into the E1 deletion in a virus vector.   In a particularly preferred embodiment of the invention, the adenovirus vector is Ad2 / C FTR-16, which is operably linked to the CMV promoter and has the deletion El Of the cystic fibrosis transmembrane regulator (CFTR) inserted into the region A gene that contains the gene and is further transduced in cells of the individual to which it is administered. It also includes a 1.6 kb deletion from the E3 region, which can promote persistent expression of offspring. Have.   If necessary, transient expression of the transgene may be, for example, in the E4 region or in it. Provides continuous expression, such as removal of specific open reading frames contained in Can be achieved using an adenovirus vector that lacks such components Will be recognized by those skilled in the art.   To produce a recombinant adenovirus vector of the invention containing a transcription unit Contains a transcription unit inserted into the adenovirus genomic fragment Plasmids are converted to a linearized viral genome derived from the adenovirus vector in question. Homology between genomic fragments and virus in recipient cells Cotransfected under conditions such that recombination occurs. Preferably, the transcription unit is E1 deleted Designed in the part (engineered). As a result, the transcript encoding the desired transgene The site where the unit is cloned into the plasmid in the adenovirus vector To make a recombinant adenovirus vector. Homologous recombination Later, as demonstrated by the formation of viral plaques, the vector genome Encapsidated into lion. Preparation of replication-defective vector stocks Cell lines in which the Rus gene has been deleted from the vector, eg, deleted adenovirus E1 This can be achieved using 293 or A549 cells containing the genomic sequence. . After plaque amplification in a suitable complementing cell line The virus was recovered by freeze-thaw followed by cesium chloride centrifugation. Can be used for purification. Alternatively, using chromatographic methods, Loose purification can be performed (eg, International Application No. PCT / US96 / 1387). No. 2, filed Aug. 30, 1996, which is incorporated herein by reference. Is).   The titer of the replication-defective adenovirus vector stock is based on the complementing cell line, For example, it can be measured by plaque formation in 293 cells. example For example, end-point dilution using an antibody against the adenovirus hexon protein (end-p oint dilution) can be used to quantify virus production (Arment ano, et al., Hum. Gene Ther. 6: 1343-1353, 1995) .   Analysis can be performed in a tissue culture system to assess the persistence of the transgene. Book Cell lines that can be infected by the adenovirus vectors of the invention include: Suitable for analysis to determine the level and duration of expression of transgenes contained is there. The transgene can encode a biologically useful protein, or Sends a transgene that allows the adenovirus vector to be expressed continuously. Code for the marker protein used to test whether can do. Suitable for assessing the persistence of expression for the desired transgene Intelligent molecular analysis can be performed, for example, by Northern blot, S1 analysis or reverse transcription polymerase. Includes measurement of transgene mRNA by chain reaction (RT-PCR). Introduction The presence of the protein encoded by the gene was determined by Western blot, immunoprecipitation. Can be detected by precipitation, immunocytochemistry, or other methods known to those of skill in the art. it can.   To assess the persistence of transgene expression using the present invention, in particular, a possible host Persistence of transgene expression over time based on the background of the immune response Animal models are appropriate for assessing gender. Such animal models are Ease of delivery of ills vectors, identification of transgenes, related molecular analysis, and And the likelihood of clinical status assessment. If the transgene encodes a biologically active protein, such The use of animal models to represent disease states that respond to protein donations has Perfect for evaluating good. For example, designed by mutant CFTR gene Adenovirus vector of the present invention using a transgenic mouse Repair of the function caused by the provision of wild-type CFTR from Japan . Jackson Laboratory (Bar Harbor, Maine) Mouse strain FABP-hCFTR (Zhou et al., Science) e 266: 1705-1708, 1994) can be used in such experiments. it can.   A suitable in which the adenoviral vector of the invention can be analyzed Such animals include, but are not limited to, mice, rabbits, rats and monkeys. Of the present invention A suitable mouse system in which adenovirus vectors can be analyzed Tori includes, but is not limited to, C3H, C57B1 / 6 (wild type and nude) and BALB / c (Taconic Farms, Germantown, New York) Hand).   If it is desirable to assess the host immune response to vector administration, Immunoreactive animals to identify the specific adverse response produced by the system And tests in immunodeficient animals can be compared. Immunodeficient animals, such as Vector performance and transgene irrespective of acquired host response using nude mice Offspring expression can be characterized to identify other determinants of transgene persistence .   After administration of the vector, the immunoreactivity is measured to determine the magnitude of the host immune response. Specific parameters of the host immune response in animal models or treated individuals. Can be measured. Measuring both humoral and cell-mediated immune processes Can be. Appropriate analysis for cell-mediated host immune response is based on the spleen of treated animals. Includes T cell proliferation analysis and CTL analysis from cells isolated from the gut (Kapla n et al., Gene Ther. 3: 117-127, 1996). Into the site of infection T cell infiltration also indicates a host immune response (Ginsberg et al., Proc. Nat. l. Acad. Sci. ScL USA 88: 1651-1655, 1991). standard Anti-adenovirus antibodies or anti-transduction in serum and other fluids using immunological methods Gene antibodies can be measured to assess host humoral response to vector administration. Wear.   A transgene in an adenovirus vector is administered to the respiratory epithelium of test animals In certain embodiments that are CFTR, suitable animal models, such as C57B1 / 6 or Is analyzed in lungs of BALB / c mice, cotton rats, or Rhesus monkeys Can be Molecular markers that can be used to measure persistence of expression Represents CFTRm by Northern blot, S1 analysis or RT-PCR, for example. Includes measuring RNA. The presence of the CFTR protein was confirmed by Western blot Detection by sedimentation, immunocytochemistry, or other methods known to those skilled in the art. Can be. The adenovirus vectors of the present invention may be used to interact with specific cell surface receptors. Modified, e.g., adenovirus fiber or penton protein To express surface molecules that promote targeting of specific cell types, such as Can be designed. Adenovirus vectors are most often infected. Other types of vectors disclosed herein, but can be introduced into cells by Adenovirus of the invention for mediating delivery, eg, delivery to cells -The use of cationic amphiphiles complexed with vectors is also within the scope of the present invention. Mosquito Thione amphiphiles have a chemical structure that includes polar and non-polar domains, Binds to biologically useful molecules and nonpolar domains cross the lipid membrane. Promote the entry of such molecules. Cationic parents preferred for such delivery The medium is described in PCT Publication No. WO 96/18372, issued on June 20, 1996. No., which is incorporated herein by reference.   To obtain specific phenotypic results using the adenovirus vectors of the invention , Any number of transgenes can be delivered to a cell and expressed.   The present invention further provides for the maintenance of a transgene encoding a biologically active protein. In order to achieve sexual expression, one or more types of cells of an individual requiring such a transgene are required. Adenovirus of the invention, which can be administered in an effective amount to deliver the above transgene The present invention also relates to a composition containing the vector.   The composition includes a physiologically acceptable carrier, including a suitable solvent. Can be As used herein, "physiologically acceptable carrier" Any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents And an absorption delaying agent. Any conventional media and agents are incompatible with the active ingredient Unless otherwise, its use in the composition of the invention is considered.   The route of administration of the composition containing the adenovirus vector may be, for example, target organ or Is direct administration to tissues, intranasal, intramuscular, subcutaneous, intradermal, oral and other parenteral administration Includes conventional and physiologically acceptable routes, such as routes.   The present invention further provides for the maintenance of a transgene encoding a biologically active protein. In order to express sexually, one type of cell is used with the adenovirus vector of the present invention. It is desirable to deliver the above transgene, in vivo or ex vivo o It also relates to the use of the composition of the invention in applications. For in vivo applications Direct administration of the adenovirus vector of the present invention prepared in a composition to the cells of an individual Need to do. Ex vivo uses were maintained in vitro Transduction of the adenovirus vector directly into the harvested autologous cells followed by transduction The cells need to be re-administered to the recipient.   For sustained expression of a transgene encoding a biologically active protein The dose of the adenovirus vector of the invention, which is administered to the individual at Conditions to be met, age, weight and clinical condition of the individual, and provision of biologically active proteins Determined in relation to a variety of parameters, including specific molecular defects that require It is. The dose is preferably as measured by molecular analysis or clinical markers , Selected such that administration results in sustained expression of the transgene and specific phenotypic results Is done. For example, an agent of the invention containing a CFTR transgene administered to an individual. To determine the persistence of expression of a transgene encoded by a denovirus vector , For example, CFTRmR by Northern blot, S1 or RT-PCR analysis Measurement of NA or Western blot, immunoprecipitation, immunocytochemistry or those skilled in the art Including measurement of CFTR protein detected by other methods known to This can be done by child analysis. Specific by CFTR transgene delivery A suitable clinical trial that can be used to evaluate phenotypic results is the P Includes FT assessment and lung radiation assessment. Delivery of transgenes encoding CFTR Demonstration of translocation has been demonstrated in cystic lines receiving vectors containing this transgene. Detecting the presence of functional chloride channels in cells of individuals with fibrosis (Zabner et al., J. Clin. Inves). t. 97: 1504-1511, 1996; U.S. Patent No. 5,670,488, Published September 23, 1997, incorporated herein by reference). other Persistence of transgene expression in various disease states is also the most relevant The same analysis can be performed using the parameters.   Sustained expression of a biologically active protein encoded by the transgene, An individual is given a transgene contained in a vector to obtain a specific phenotypic result. The dose of the adenovirus vector of the present invention that can be used to obtain About 10 for human⁸10 from the infectious unit (IU)¹¹I. U . Range.   Formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage It is particularly advantageous to do so. As used herein, dosage unit forms should be treated. Means a physically discrete unit suitable as a unit dose for a subject, with each unit being Calculated to produce a specific phenotypic result, along with the required physiological carriers Contains a predetermined amount of active ingredient. The specifications of the novel dosage unit form of the present invention Characteristics of adenovirus vectors used and limitations inherent in the art of preparation And is directly dependent on them. Main active ingredients (adenovirus • Vectors) are physiologically acceptable carriers for convenient and effective administration Together with the dosage unit form as described above.   The adenovirus vector of the invention for supplying one or more transgenes to an individual. The greatest benefit arising from the administration of a vector and achieving specific phenotypic results May need. Such multiple doses can be performed with the same adenovirus vector Or alters viral antigen presentation and weakens the host immune response A different vector designed to contain the same transgene except for rotation to control May require the use of a reactor.   The practice of the present invention is within the skill of the art unless otherwise specified. Protein chemistry, molecular virology, microbiology, recombinant DNA technology and medicine Use conventional methods of pharmacy. Such methods are explained fully in the literature. For example,Current Protocols in Molecular B iologv, Ausubel et al., Edited by John Wiley & Sons , New York, 1995;Remington's Pharmaceuti cal Sciences, 17th edition, Mack Publishing Co. . Easton, PA. See 1985.   The present invention is further described by the following specific examples, which In no sense, it is not intended to limit the scope of the invention. Example 1: Adenovirus vector   Plasmids pAd/E4+/E3@1.6 (FIG. 1) and Ad2 / CFTR-5 Recombination by homologous recombination with viral DNA isolated from the virus The adenovirus vector Ad2 / CFTR-15 was constructed. Ad2 / CF TR-5 is a CMV promoter (nucleotides -523 to -14, Boshar et al., Cell 41: 521-530, 1985). As human CFTR (hCFTR) and BGH inserted into the deletion site of the E1 region Contains polyA signal. The E2 and E3 regions of Ad2 / CFTR-5 are wild-type Contains the denovirus serotype 2 sequence (Ad2) and all E4 sequences are E4O Deleted except for RF6. Plasmid pAd / E4 + / E3 △ 1.6 From the SpeI site to the right inverted terminal repeat (ITR) in nucleotide 27123 And has the right-hand end of adenovirus 2. The E3 region has a 1549 bp deletion (Ad 2 nucleotides 29292-30840).   Ad2 / CFTR-5 DNA is converted to the unique P at Ad2 nucleotide position 28612. Cleavage at the acI site. At a unique ClaI site directly downstream of the ITR And cut to linearize plasmid pAd / E4 + / E3 △ 1.6. did. Using these two DNAs, CaPO_Four293 cells by sedimentation Simultaneously populated. Virus plaques are isolated and developed, and the recombinant virus is Identified by restriction digestion of total DNA from Rus infected cells.   The Ad2 / CFTR-16 virus derived from this recombination has a wild-type E2 region and an E2 It has three regions (FIG. 2) and has a 1549 bp E3 deletion. Degree of modification of E3 region Is shown in FIG.   The configuration of Ad2 / CFTR-2 has already been described (Kaplan et al., Gen. e Ther. 3: 117-127, 1996). Ad2 / CFTR-2 is PG HCFTR as a transgene driven by the K promoter and wild-type ( w. t. ) Comprising the E2 and E3 regions, the E4 transcription unit being the open reading of E4 Frame 6 (ORF6). Example 2: Sustained transgene expression in immunoreactive and nude miceMethod   C57B1 / 6 (wild type and nude) and BALB / c mice were Taconic   Farms (Germantown, NY) About 2x10 depending on the road⁹Using the infectious unit (IU) the vector Ad2 / CFT R-16 was added dropwise. RNA was isolated as described below and purified from human CFTR (hC FTR) levels of expression were measured at various time points by quantitative RT-PCR Was.   A tissue sample from the lung of each mouse was used as an RNA Stat-60 solution (Tel-T est B, Inc). RNA is Mini-Beadbe ater (Biospec Corp., Bartlesville, OK) Acid guanidinium / phenol-chloroform method (Chomczynski et al. Anal. Biochem. 162: 156-159, 1987) did. RNA samples were pooled at each time point for each group of animals.   Total RNA (7.5 mg) was treated with DNase. In addition to the sample RNA, 10^ThreeOr 10^TwoSynthetic competitive RNA of molecule (469b having CFTR sequence at both ends) pCAT transcript) was added to the reaction mix. Invitrogen (San Reverse transcription was performed with a cDNA kit from Diego, CA). After reverse transcription , CDNA from each test group was analyzed using the conditions and reagent concentrations as described above for the PCR reaction. Was used to amplify. The PCR product from the hCFTR sequence is 544 bp; The competitor product is 469 bp. The product intensity ratio should be adjusted to the appropriate copy number Logarithmic concentration of Ad2 / CFTR-8 DNA run with body DNA (Hehir J. et al. Virol. 70: 8459-8467, 1996) (10¹-10⁶) Gene expression was quantified by comparison with the intensity ratio of a DNA standard curve containingresult   As shown in FIG. 4, Ad2 / CFTR-16 was compared to nude C57B1 / 6 mice. HCFTR retention for up to 30 days in immunoreactive C57B1 / 6 mice This resulted in persistent expression. Next, hCFTR mRNA expression was measured at intervals up to 70 days. Measured. As shown in FIG. 5, Ad2 / CFTR-16 is immunoreactive C HCFTR expression occurred on day 70 in 57B1 / 6 mice, which was 3 It did not differ significantly from the expression measured on day.   In immunoreactive BALB / c mice, Ad2 / CFTR-16 The resulting sustained expression of hCFTR was observed up to day 45 (FIG. 6) and by day 70 Persistence has been further observed (Figure 7). In the lungs of immunoreactive animals Such sustained expression of hCFTR in the first generation (E2⁺, E4⁺Adenow Not observed in previous studies with irs vector for more than 2-3 weeks .   Ad2 / CFTR-16 expresses hCFTR in C3H mice at day 70 Which did not differ significantly from the expression measured on day 3 (FIG. 8). Example 3: Sustained transgene expression compared to endogenous geneMethod   Further using the alternative RNA deletion method, the results obtained by the quantitative RT-PCR method were used. Demonstrated fruit. This method uses the hCFTRmR derived from the Ad / CFTR vector. NA levels are assessed relative to endogenous murine CFTR mRNA. RT-PCR Increased both murine CFTR mRNA and Ad vector-derived hCFTR mRNA. Performed on RNA isolated from lung tissue using primers to span. Next Next, the RT-PCR product was digested with MspI, which cuts only hCFTR. And analyzed by gel electrophoresis.result   As shown in FIG. 9, murine CFTR mRNA was present at steady levels throughout the experiment. And served as an internal control for the RT-PCR reaction. Ad2 / CFTR- The levels of 16-encoded hCFTR mRNA were low over the time course of the experiment. It did not change significantly. However, hCFTRmRN from Ad2 / CFTR-5 A expression had fallen to background levels by day 45. This method is Although not quantitative, hC expressed from Ad2 / CFTR-16 in mouse lung FTR mRNA levels were measured over the time course of this experiment (70 days). It appears to be above the level of mouse CFTR mRNA expression. Example 4: Ad2 / CFTR-16 inheritance in nasal epithelium of CFTR mutant mice Persistence of offspringMethod   To determine whether Ad2 / CFTR-16 corrects CF chloride secretion deficiency For this purpose, a gene transfer experiment was performed in the nasal cavity of a mouse deficient in CFTR activity. The mice used in these experiments had a knockout deletion in the endogenous CFTR gene. However, for hCFTR cDNA controlled by an intestinal specific promoter Is transgenic (Zhou et al., Science 266: 1705). １７０1708, 1994). This gene composition allows animals to enter the respiratory tract, including the nasal epithelium. Is functionally defective with respect to CFTR. 10⁹IU Ad2 / CFTR- 16 was administered to the nasal mucosa of these mice as previously described (Jiang et al., Hum. Gene Ther. 8: 671-680, 1997). Briefly explain The mice were then treated with 2,2,2-tribromoethanol (0. 0,0%) in 0.9% NaCl. Intraperitoneal injection with 4 mg / g) and t-amyl alcohol (0.4 μl / g) Anesthetized. A catheter connected to a 1 ml syringe attached to a microperfusion pump Contain Ad2 / CFTR-16 using Tell (pull from PE20 tubing) Solution (10⁹IU in 100 μl of PBS) at a rate of 1.6 μl / min into the nasal cavity. Let it flow. With continuous perfusion, the mouse nasal epithelium was adenoviral for 1 hour. And was constantly exposed to S.vector. PD crossing nasal epithelium of CF mouse described (Crubb et al., Amer. J. Physiol. 266: C1478-C1483, 1994; Jiang et al., Hum. Gene The r. 8: 671-680, 1997; Zeiher et al. Clin. Inve st. 96: 2051-2064, 1995).result   FIG. 10a shows that the initial basal potential difference (PD) is untreated double transgenic CF ( − / −) Mice are significantly more negative than wild type mice. Show. Perfusion of nasal epithelium with Ringer's solution was compared with wild-type (+ / +) -) Both mice resulted in a decrease in basal PD. Contains amyloid (100 μM) Subsequent perfusion with Ringer's solution further reduced PD in all groups . This decrease is more pronounced in CF (-/-) mice than in wild-type (+ / +) mice. Was significantly larger.   In untreated double transgenic CF (-/-) mice, amyloid (10 0 μM) in the presence of NaCl and Na gluconate (Low Cl) in Ringer's solution. ) Is small, as previously observed in CF null mice. Poles were induced (FIG. 10b) (Crubb et al., Amer. J. Physiol. 2). 66: C1478-C1483, 1994: Jiang et al., Hum. Gene Ther. 8: 671-680, 1997; Zeiher et al. Clin. I nvest. 96: 2051-2064, 1995). These results are The electrophysiological properties of the nasal epithelium of transgenic CF mice indicate that CF null and △ Suggests similarities to the same properties of F508 mice (Crubb et al., Amer. J. Physiol. 266: C1478-C1483, 1994; Jiang Hum. Gene Ther. 8: 671-680, 1997; Zehei r et al. Clin. Invest. 96: 2051-2064, 1995). Administration of Ad2 / CFTR-16 reduces basal PD (FIG. 10a) and reduces Cl Repairs the hyperpolarization of the response to (Fig. 10b), which indicates that functional CF within the nasal epithelium Demonstrated the presence of TR. Moreover, these electrophysiological changes were tested It did not decrease significantly even on the 15th day after vector administration, which was the longest time. This In contrast, previous experiments were induced by administration of Ad2 / CFTR-5. Electrolyte transfer was reduced within one week (Jiang et al., Hu). m. Gene Ther. 8: 671-680, 1997). These data are In the nasal epithelium of CF mice, Ad2 / CFTR-16 was converted to Ad2 / CFT. FIG. 9 shows that it produces greater sustained expression of functional CFTR as compared to R-5 Hinting. Example 5: To immunoreactive mice treated with Ad2 / CFTR-16 Presents the anti-Ad vector CTL.Method   Longevity of expression from Ad2 / CFTR-16 elicits a CTL response, Is it explained by the unexpected failure to cause escape from the visual system? To study the CTL response after intranasal administration of Ad2 / CFTR-16 to test did.   The detailed protocol of the CTL analysis is essentially as previously described (Ka plan et al., Hum. Gene Ther. 8: 1095 to 1104, 1997 Scaria et al., Gene Ther. 4: 611-617, 1997; Wa dworth et al. Virol. 71: 5189-5196, 1997). Simple Briefly, spleen cells from animals from the same group were pooled and 100 multiple infections ( (MOI) by syngeneic fibroblasts infected by Ad2 / CFTR-16 Stimulation was performed in vitro. After 5-7 days of culture, the cytolytic activity is determined. Was analyzed. Target fibroblasts were treated with Ad2 / CFTR-16 at 100 MOI. Γ-interferon (Genzyme, Camb). r.m., MA) for about 24 hours and then MHC Class I It was used to enhance expression and antigen presentation to effector CTL. This fibroblast Cells⁵¹chromium(⁵¹Cr, NEN)   Overnight (30 μCi / 10^FiveCells) in the holes of a 96-well round bottom plate 100 μl (5 × 10^ThreeFibroblasts / well). 10 effector cells 0 μl volumes were added in triplicate at various effector: target cell ratios. 37 ° C / 5% CO_Two20 μl of cells after 5 h incubation in Unretained supernatant was collected from each well and Microbeta Trilux liquid scintillator was collected. In a filtration counter (Wallac, Gaithersburg, MD) I counted. % Lysis was calculated as follows: result   Vector-specific CTL responses in C57B1 / 6 and BALB / c mice Observed (FIG. 11). No CTL response specific for hCFTR has been demonstrated. Example 6: Repeated administration of Ad2 / CFTR-16Method   The CTL response to Ad vectors expressing weak immunogenic transgenes was After a single administration of the vector to the lung, as previously demonstrated for administration to Invalid (Wadworth et al., J. Virol. 71: 5189-5176). , 1997). Important issues regarding the effective application of gene therapy for the treatment of CF Is the immune response after repeated administration. Effective repeated administration of an Ad vector is, for example, Antibodies to CD40 ligand (Scaria et al., Gene Ther. 4: 6 11-617, 1997) or deoxyspergualin (Kaplan et al., Hu). m. Gene Ther. 8: 1095-1104, 1997). It can be achieved by the use of economies. The use of such agents will increase the neutralizing antibody response. Not only weaken and allow for repeated gene transfer, but also Confuses research on the nature of the alveolar immune response. To avoid these problems , BALB / c mice were initially dosed (5 × 10⁸IU) Ad2 / CFTR-16 or Was derived from an empty Ad vector lacking the hCFTR cDNA (Ad2 / EV). Treatment on day 0 and then on day 14 by the dose of Ad2 / CFTR-16. Challenged.result   During the course of the experiment, the CTL response to the vector antigen is stimulated, but the neutralizing antibody is The suppression level is not reached. At day 0, 10 parallel groups of animals⁹IU A The vector-derived hCFTR mRNA level was treated with d2 / CFTR-16. Was measured until day 59 (FIG. 12). These are Ad2 / CFTR-16 or Pre-exposure of the animal to either Ad2 / EV can reduce the single vector dose. Compared to the challenge dose of Ad2 / CFTR-16 compared to animals that received It did not demonstrate any effect on expression duration.

────────────────────────────────────────────────── ─── Continuation of front page    (72) Inventors Wadsworth, Samuel, See.             United States Massachusetts, S.             Rusbury, Straw Hollow Lane             Ten

Claims (1)

[Claims]   1. Lack of the E1 region and one or more open reading frames of the E3 region Adenovirus vector containing the missing adenovirus genome.   2. The gene encoding the E3gp19K protein is contained in a vector. The vector according to claim 1, wherein   3. The gene encoding the E3 11.6K protein is deleted. 2. The vector according to 1.   4. The gene encoding protein IX is located at the normal position in the adenovirus genome. The vector according to claim 1, wherein the vector is rearranged at a position other than the position.   5. 2. The method of claim 1, wherein the transgene has been inserted into a deletion in the E1 region. vector.   6. The vector of claim 5, wherein the transgene is operably linked to an expression control sequence. Tar.   7. Expression control sequences include the cytomegalovirus immediate-early promoter. The vector according to claim 6.   8. 8. The transgene encodes a cystic fibrosis transmembrane regulatory protein. The described vector.   9. The vector according to claim 8, which is Ad2 / CFTR-16.   10. An adenovirus vector according to claim 5 and a physiologically acceptable carrier. A composition comprising a rear.   11. An adenovirus vector according to claim 9 and a physiologically acceptable carrier. A composition comprising a rear.   12. Continuously expressing the transgene in cells of an individual in need of the transgene A method, comprising: an effective amount for such sustained expression of a transgene in said individual. A method comprising administering the composition of claim 10.   13. An individual in need of the cystic fibrosis transmembrane regulatory transgene A method for continuously expressing a transgene in the above-described cells, 12. A method comprising administering an effective amount of the composition of claim 11 for expression.